Several studies have shown that thermal straightening has an influence on the concentration of THC, ethyl glucuronide and cocaine in hair. Heat treatment of hair may decrease the concentration of cocaine in hair and increase BZE and cocaethylene. A BZE/cocaine ratio greater than 1 was proposed to identify thermal straightening prior to sampling [1] . The aim of this study was to evaluate the influence of thermal straightening on other metabolites of cocaine: ecgonine methyl ester (EME), norcocaine and on the marker of crack use: anhydroegonine methyl ester (AEME). Forty-one capillary samples positive for cocaine were treated in vitro with iron plates heated to 200 °C. One lock of hair was put in contact 30 times for 2 seconds with a hair straightener, the other lock was not treated. After decontamination and pulverization, both hair locks were incubated in a phosphate buffer for 2 h in an ultrasonic bath. Analytes with their deuterated analogues were isolated using solid phase extraction and then derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatized products were determined by GC-MS in EI mode. Hair concentrations of cocaine, BZE, EME, cocaethylene, norcocaine and AEME were compared with those in the corresponding untreated strands. After treatment, a median decrease of concentrations was observed for cocaine (−55.0%), EME (−28.2%), cocaethylene (−41.6%) and norcocaine (−43.2%), whereas a median increase was found for BZE (+28.6%) and AEME (+122%). The decreases of cocaine, EME, cocaethylene and norcocaine concentrations after treatment with a heating iron are most likely due to thermal denaturation of the mentioned substances. The increased concentrations of BZE and AEME could be explained by chemical transformation of cocaine which is incorporated in the hair matrix into BZE and AEME. Our results show that presence of AEME in hair does not prove that crack was consumed. A crack use should only be considered in case where thermal straightening before sampling may be excluded (BZE/cocaine ratio lower 1). Regarding our in vitro results, thermal straightening should be documented during hair collection and should be considered for interpretation of AEME results in hair.